JP2010520336A - Benzophenone or benzoic acid anilide derivatives containing carboxyl groups as enzyme stabilizers - Google Patents

Abstract

The present application relates to detergents and detergents containing a benzophenone or benzoylanilide derivative containing a carboxyl group that function as protease inhibitors and are suitable as enzyme stabilizers. A further problem is the use of such compounds as reversible inhibitors of proteases, their use as stabilizers for detergent or detergent formulations, and further methods and uses therefor.

Description

  The present invention relates to a detergent or detergent containing a carboxyl group and acting as a protease inhibitor and thus containing a suitable enzyme stabilizer, benzophenone or a benzoic acid anilide derivative.

  The use of enzymes in cleaning and cleaning agents has been established in the prior art. Enzymes serve to expand the range of performance of each drug by their specific activity. These include in particular hydrolases such as proteases, amylases, lipases and cellulases. The first three enzymes mentioned above contribute directly to soil removal as a result of hydrolyzing protein, starch and fat. Cellulases are particularly used because of their tissue effects. Another group of detergent and detergent enzymes are oxidases, especially oxidases, which are preferably used in combination with other ingredients as needed to bleach the soil or create the bleach itself. It is done. In addition to these enzymes that are subject to persistent optimization, additional enzymes used in cleaning and cleaning agents are constantly available so that, for example, optimal approaches to certain types of soils can be made available, in particular pectinase, β -Other hemicellulases for the hydrolysis of glucanases, mannases or especially certain plant-derived polymers are available.

  Proteases are enzymes that have been established for a long time and are actually included in all modern effective detergents and detergents, in particular serine proteases and also subtilases. They cause the degradation of proteinaceous soils that are washed. However, they are also subject to autohydrolysis (self-proteolysis) as well as hydrolyzing all other proteins (ie especially other enzymes) contained in each drug. This occurs especially during the washing process, i.e. in aqueous washing solutions, when relatively good reaction conditions are widespread. However, this also occurs during storage of each drug, causing a certain loss of enzyme activity (eg, protease activity) and is also associated with a long storage period. In general, the enzyme activity in a detergent or detergent is inversely proportional to the storage period, with a decrease in enzyme activity due to a longer storage period. This is particularly a problem in gel or liquid formulations containing water, since both the reaction medium and the hydrolysis reagent can be used with the water contained in the formulation.

  Thus, one goal in the development of detergents and detergents is to stabilize the enzymes contained in the drug during storage in particular. This is understood to refer to protection against various undesirable effects such as denaturation or degradation due to physical action or oxidation. One focus of these developments consists of protection against proteolytic cleavage of proteins and / or enzymes contained in the drug. This can be achieved, for example, by creating a physical barrier by encapsulating the enzyme in specific enzyme granules or finishing the agent in two-chamber or multi-chamber systems. . Another method that is often used consists of the fact that proteases are added to the drug as compounds that inhibit proteases and thus act as stabilizers for proteases and other proteins and enzymes contained in the drug as a whole. These proteases must be reversible protease inhibitors, as protease activity should be temporarily suppressed, especially during storage, but not during the washing process.

  Reversible protease inhibitors known in the prior art include polyols, in particular glycerol and 1,2-propylene glycol, benzamidine hydrochloride, borax, boric acid, boronic acid or salts or esters thereof. Of particular mention should be made of derivatives of aromatic groups, such as ortho-, meta- or para-substituted phenylboronic acids, in particular 4-formylphenylboronic acid (4-FPBA) and / or salts or esters of the aforementioned compounds. . Particularly good protection is obtained when boric acid derivatives are used with polyols because they can form complexes that stabilize the enzyme. Peptide aldehydes, ie oligopeptides with a reduced C-terminus, in particular 2-5 monomeric oligopeptides, have been described for this purpose. In particular, reversible protease inhibitors include ovomucoid and leupeptin. Specific reversible peptide inhibitors as well as protease fusion proteins and specific peptide inhibitors are used for this purpose.

  However, polyols such as glycerol and 1,2-propylene glycol have been found to be unfavorable because they require high use concentrations so that other active ingredients of each drug can only be present in a small proportion.

  Of the serine protease inhibitors that are effective at relatively low concentrations, boric acid derivatives take the leading position. For example, international patent application WO 96/21716 A1 discloses that boric acid derivatives that act as protease inhibitors are also suitable for the stabilization of enzymes in detergents and detergents. A particularly effective selection of stabilizers is disclosed in international patent application WO 96/41859 A1.

International Publication No. 96/21716 Pamphlet International Publication No. 96/41859 Pamphlet

  However, despite their stabilizing effects, boric acid derivatives have significant disadvantages. Many boric acid derivatives, such as borates, form undesirable by-products with several other detergents and / or detergent ingredients, which can no longer be used for preferred cleaning purposes in each drug and have been washed Things can even remain behind as impurities.

  The resulting purpose is therefore to identify suitable boron-free compounds as enzyme inhibitors in detergents and detergents that act as protease inhibitors and as a result.

  Of particular interest here is the use of detergents and detergents (including in particular those containing water) which are generally liquid, gelled or pasty.

  This object is achieved by the following drugs.

［式中、
（ａ）Ｘはカルボニル基（Ｃ＝Ｏ）または酸アミド基（ＮＨＣＯ）を表し、
（ｂ）Ｒ１、Ｒ２、Ｒ３、Ｒ４およびＲ５（環１における）は、少なくとも１つのカルボキシル基（ＣＯＯＨ）が該環中に存在するように、水素（Ｈ）、カルボキシル基（ＣＯＯＨ）、メチル基（ＣＨ_３）、エチル基（Ｃ_２Ｈ_５）、ヒドロキシル基（ＯＨ）、ヒドロキシメチル基（ＣＨ_２ＯＨ）、アミノ基（ＮＨ_２）および／またはハロゲンを表し、
（ｃ）Ｒ６、Ｒ７、Ｒ８、Ｒ９およびＲ１０（環２における）は、少なくとも１つのカルボキシル基（ＣＯＯＨ）が該環中に存在するように、水素（Ｈ）、カルボキシル基（ＣＯＯＨ）、メチル基（ＣＨ_３）、エチル基（Ｃ_２Ｈ_５）、ヒドロキシル基（ＯＨ）、ヒドロキシメチル基（ＣＨ_２ＯＨ）、アミノ基（ＮＨ_２）および／またはハロゲンを表し、かつ、
（ｄ）場合により、Ｒ１〜Ｒ１０の２つの基（Ａ）および（Ｂ）は互いにオルト位にあってよく、ここで（Ａ）は、（ｂ）および／または（ｃ）に記載の必須のカルボキシル基（ＣＯＯＨ）または場合により付加的なカルボキシル基であり、かつ（Ｂ）はヒドロキシメチル基であり、それらは上記の基としてまたは場合によりＣＨ_２−Ｏ−ＣＯ−基として存在し、それらを含む環の炭素原子とともに五員環ラクトンを表す。］ A detergent or detergent containing a protease and a compound having the following general structural formula.

[Where:
(A) X represents a carbonyl group (C═O) or an acid amide group (NHCO),
(B) R1, R2, R3, R4 and R5 (in ring 1) are hydrogen (H), carboxyl group (COOH), methyl group so that at least one carboxyl group (COOH) is present in the ring; (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen,
(C) R6, R7, R8, R9 and R10 (in ring 2) are hydrogen (H), carboxyl group (COOH), methyl group such that at least one carboxyl group (COOH) is present in the ring (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen, and
(D) Optionally, the two groups R1 to R10 (A) and (B) may be ortho to each other, wherein (A) is an essential component as described in (b) and / or (c) A carboxyl group (COOH) or optionally an additional carboxyl group, and (B) is a hydroxymethyl group, which are present as the above groups or optionally as CH ₂ —O—CO— groups, Represents a five-membered lactone with the carbon atoms of the ring it contains. ]

  According to the invention, a detergent or cleaning agent is understood to include all agents which are particularly suitable for cleaning or cleaning textile products and / or solid surfaces. Ingredients suitable for this purpose are described in detail below.

  In accordance with the present invention, proteases are understood to include all enzymes capable of hydrolyzing protein acid amide bonds. The protease is also described in detail below.

  The compound represented by the general structural formula is an aromatic compound having two benzene rings bonded by a keto group or an acid amide group according to the feature (a). This is therefore a benzophenone derivative or a benzoic acid anilide derivative.

This benzophenone derivative is in accordance with features (b) and (c) with hydrogen (as in groups R1, R2, R3, R4 and R5 (in ring 1) and / or R6, R7, R8, R9 and R10 (in ring 2) H), carboxyl group (COOH), methyl group (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen Have It is a prerequisite that at least one carboxyl group (COOH) is present on each of the two rings.

The same is true for benzoic acid anilide derivatives. Here, ring 1 is a ring that can be attributed to benzoic acid and / or its substituted product, and ring 2 is a ring that can be attributed to aniline and / or its substituted product. Can do. This benzoic acid anilide derivative is also hydrogen (H), carboxyl group (COOH), methyl group (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino The groups (NH ₂ ) and / or halogens can be present as R1, R2, R3, R4 and R5 (in ring 1) and / or R6, R7, R8, R9 and R10 (in ring 2). In this case also, it is an essential condition that at least one carboxyl group (COOH) is present in each of the two rings.

According to the feature (d), when having two groups of R1 to R10 as the substitutable groups in one of the two rings 1 or 2 as (A) and (B) in the ortho position to each other, benzophenone or benzoic acid Acid anilide derivatives are also relevant to the present invention. (A) is an essential carboxyl group (COOH) or any other carboxyl group as described above in (b) and / or (c), and (B) is a hydroxymethyl group; Such groups or optionally the group CH ₂ —O—CO are present and together with the carbon atoms of the ring containing them form a 5-membered lactone.

  The two substituents (A) and (B), which can be carboxyl groups (COOH) and / or hydroxymethyl groups according to (b) and (c), can also be adjacent to each other within one ring . That is, they are ortho to each other and exist in the form of a hydroxyl group and a carboxymethyl group at the same time. In this case, these two groups together form a lactone and may be bound in the form of a lactone to the protease being inhibited. Also, conjugation can occur without prior changes in the lactone form. For the practice of the present invention, it is also possible to identify the form of the lactone synthetically and add the lactone already formed as a stabilizer for each drug. The most appropriate form is to be determined empirically based on the protease to be inhibited and the intended stabilizer, which should not pose any fundamental difficulty to those skilled in the art.

  According to a preferred embodiment based on features (b) and (c) and the number of carboxyl groups per molecule, when counting carboxyl groups, this lactone carboxyl group is the carboxyl group of features (b) and / or (c) May be present in addition to other carboxyl groups.

  In another preferred embodiment of the invention, a thiophene ring may be present instead of one of the two benzene rings. The arrangement of the substitutable groups 1 ′, 2 ′, 3 ′ therein can be analogized with the references made for the above features (b), (c) and (d).

  This embodiment allows similar non-covalent interactions with the protease to be inhibited, or even better non-covalent interactions in each case via the aromatic thiophene ring. Furthermore, the sulfur atoms contained therein allow further interaction, especially via free electron pairs, which can be advantageous in individual cases. These particularly preferred compounds are those in which dynamic parameters based on the specific protease to be inhibited have been studied and optimized through optimal selection and substitution configuration. Here, it is possible to rely on techniques obtained on the basis of compounds having a benzene ring.

The present invention comprises the aforementioned compounds in fully protonated and / or deprotonated form. In particular, carboxyl groups (COOH) and optionally amino groups (NH ₂ ) are present as carboxylate groups (COO ⁻ ) and / or ammonium groups (NH ₃ ⁺ ), depending on the pH of the surrounding medium. If desired, reversely charged cations (H ⁺ , Na ⁺ , K ⁺ etc.) and / or anions (Cl ⁻ , Br ⁻ , formate, acetate etc.) may also be present. The present invention can embody all these forms. According to the invention, the decisive factor is the respective interaction between the compound according to the invention and the protease to be inhibited / stabilised.

Without being limited to this theory, according to the present invention, it is believed that the compounds related to the present invention form a complex with the inhibited / stabilized protease of the present invention. This complex appears to incorporate a compound according to the present invention in a substrate that is bound to a well of a protease that binds non-covalently. Thus, the active center of the protease is blocked by a compound that cannot be hydrolyzed by this enzyme, and cannot be used for hydrolysis of other proteins present. This includes reversible binding, ie an equilibrium between association and dissociation. The equilibrium factor for this reaction is indicated by the inhibition constant or K _i .

  The first advantage of the compounds according to the invention compared to the prior art is that they have a favorable inhibition constant for proteases that can be used in detergents and detergents, in addition to being able to be in lower amounts compared to polyols. Consists of the fact that This can be said for example for serine proteases and also for metalloproteases. Thus, the inhibitor binds reversibly. That is, they do not produce a temporary interaction with an enzyme that is neither too fixed nor too loose. Thus, during storage, most proteases according to the present invention exist in the form of protease inhibitor complexes. The proteases contained in them and any other proteins, in particular other enzymes, are thus protected from proteolysis by this enzyme (stabilization against proteolysis). On the other hand, when the drug of the present invention is diluted with water to prepare an aqueous washing solution or cleaning solution in the cleaning process, the binding equilibrium is shifted in the direction of dissociation, so that the complex is decomposed and most proteases related to the present invention are protein It will have degradation activity. Thus, the compounds related to the present invention are protease inhibitors that function according to the prescribed purpose, and enzyme stabilizers for detergents and detergents.

  A second advantage of the compounds according to the invention compared to the prior art is due to the fact that they have, as elements, only C, H, N and O and optionally halogen and / or sulfur and in particular no boron. Become. Thus, they do not form undesirable by-products due to other cleaning or detergent components that can be attributed to boron.

  Furthermore, in particular due to the carboxyl groups present in each aromatic ring, they are readily soluble in water and can therefore be easily incorporated into the corresponding drug, preventing precipitation during storage.

  In principle, the above compounds probably serve as reversible inhibitors because they are structurally consistent with protease substrates, especially with respect to hydrolyzed acid amide bonds. On the contrary, basically all proteases can be naturally inhibited by the compounds according to the invention, so they are suitable as protease inhibitors according to the invention. As shown on the basis of the examples of this patent application, including the advantageous effects of the experimental compounds described on the basis of serine proteases, in particular subtilases, and more particularly subtilisins based on subtilisin variants from Bacillus lentus DSM 5483, This is especially true for serine proteases.

Additional problems of the present invention relate to the following.
-The use of said compounds as reversible inhibitors and / or stabilizers of proteases within the scope of detergent or detergent formulations;
-A washing or cleaning method wherein the activated compound inhibits and / or stabilizes the protease;
The use of the detergents or detergents according to the invention for the cleaning and / or cleaning of textiles and / or hard surfaces; and the use of proteases and said compounds for the preparation of detergents or detergents.

  In all embodiments of the invention, stabilizing compounds selected from one of the following stabilizers are particularly preferred.

According to the invention, cleaning agents or cleaning agents which are stabilizing compounds having an inhibition constant (K _i ) of 0.01 to 10 mM, preferably 0.1 to 5, particularly preferably 0.5 to 2 are preferred. .

The inhibition constant K _i can be measured by the following method.

For the characterization of reversible inhibitors of enzyme activity, the inhibition constant K _i is a characteristic and important property. K _i represents the equilibrium between enzyme, inhibitor and enzyme-inhibitor complex for reversible binding. The enzyme-inhibitor complex is not catalytically active and inhibits the reaction by reducing the free enzyme concentration, which can be further utilized for substrate binding. K _i is defined by:
K _i = [I] × [E] / [EI]
In the formula, [E], [I] and [EI] are molar equilibrium concentrations of enzyme (E), inhibitor (I) and enzyme-inhibitor complex (EI), respectively. According to this definition, a substrate with a low K _i under each test condition is a good inhibitor.

式中、Ｅ：酵素
Ｓ：基質
ＥＳ：酵素−基質複合体
Ｐ：生成物
ｋ_１、ｋ_−１、ｋ_２：速度定数 K _i is measured based on the protease activity test in the presence of the corresponding inhibitor. Enzymatic parameters K _m and k _cat are well known to those skilled in the art, and Michaelis-Menten kinetics established in the prior art (Leonor Michaelis, Maud Menten (1913): The Kinetics of the Invertine Effect, Biochem. Z. 49. : 333-369) in the presence of various concentrations of inhibitors. In simple terms, the following equation applies to Michaelis-Menten kinetics:

In the formula, E: enzyme S: substrate ES: enzyme-substrate complex P: product k ₁ , k ₋₁ , k ₂ : rate constant

In this equation, k ₂ is a measure of the maximum reaction rate at substrate saturation (V _max ) and is the turnover number or molecular activity or k _cat (k _cat = V _max / [E ₀ ], where [E ₀ ] Is also known as the starting enzyme concentration). Thus, the Michaelis constant (ie, conversion rate, ie general substrate concentration at half-saturation where v = V _max / 2) is obtained as follows:
K _m = k ₋₁ / k ₁ (in the case of Michaelis-Menten, applies when k ₂ << k ₁ ) or, more generally, K _m = (k ₋₁ + k ₂ ) / k ₁ (in the case of Brigg-Hardane) , Applied when k ₂ is negligible with respect to k ₁ ).

式中、ｖ：Ｐの形成速度（ｖ＝「速度」）（ｍｏｌ・Ｌ^−１・ｓ^−１）
Ｖ_ｍａｘ：最大速度（ｍｏｌ・Ｌ^−１・ｓ^−１）
Ｋ_ｍ：ミカエリス−メンテン定数（ｍｏｌ・Ｌ^−１）
［Ｓ］：基質濃度（ｍｏｌ・Ｌ^−１） The saturation function of “Michaelis-Menten enzyme” is obtained using the parameters K _m and V _max as follows:

In the formula, v: P formation rate (v = “rate”) (mol·L ⁻¹ · s ⁻¹ )
V _max : Maximum speed (mol·L ⁻¹ · s ⁻¹ )
K _m : Michaelis-Menten constant (mol·L ⁻¹ )
[S]: Substrate concentration (mol·L ⁻¹ )

The inhibition constant K _i is obtained by measuring the initial catalyst rate (v _anf ) (the initial hydrolysis rate for the protease) by fitting the experimental data to Equation 1 below at various substrate concentrations [S]:
Formula 1: v _anf = k _cat × [S] × E ₀ / (K _m × (1+ [I] / K _i ) + S)
In the formula, [I] again represents the inhibitor concentration.

K _i may also be measured using the Cheng-Prusoff equation based on IC ₅₀ (Formula 2, Y. Cheng, WH Prusoof (1973), Biochem. Pharmacol. 22, 3099-3108). IC ₅₀ values are determined by measuring the catalytic activity of the substrate in the presence of various concentrations of inhibitor and adjusting the experimental data to a sigmoidal dose effect equation with a slope change (pseudo hill slope). This is the inhibitor concentration required to achieve 50% inhibition.

Therefore, K _i can be obtained from Equation 2 below.
Formula 2: K _i = IC ₅₀ / (1+ [S] / K _d )
In the formula, [S] represents the substrate concentration in the test, and K _d represents the dissociation constant of the substrate that can be equated to K _m for the substrate assuming that the IC ₅₀ concentration of the inhibitor matches.

The _Ki value determined in this way characterizes the compound involved in the enzyme used. In Example 1, the residual activity of the protease, ie Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1), is measured in the presence of an inhibitor. Since this is a typical subtilisin protease, the value obtained with this enzyme is also a value specific to other serine proteases, especially other subtilisin proteases. In case of doubt, the exact value for the protease of interest must be determined based on each specific enzyme.

  In the cleaning or cleaning agent of the present invention, which is present mainly in solid form in a preferred embodiment and predominantly in liquid, paste or gel form in the second embodiment, the protease is 2 μg to 20 mg per gram of drug, preferably Is contained in an amount of 5 μg to 17.5 mg / g of drug, particularly preferably 20 μg to 15 mg / g of drug, most particularly preferably 50 μg to 10 μg / g of drug.

Stabilizers, in particular for the drugs of the invention, in an amount of up to 50 mg / g of drug, preferably in an amount of up to 10 mg, particularly preferably in an amount of up to 7 mg, most particularly preferably in an amount of up to 5 mg / g of drug. Exists. Furthermore, the stabilizer is present in an amount of 0.01 to 100 × K _i (based on the protease present), preferably 0.1 to 10 × K _i , particularly preferably 1 to 5 × K _i. Preferred for.

  The molar ratio of stabilizer to protease is preferably 1: 1 to 1000: 1, in particular 1: 1 to 500: 1, particularly preferably 1: 1 to 100: 1, most particularly preferably 1: 1 to 20: 1.

  In addition to the stabilizers according to the general structural formula above, the agents of the present invention may contain at least one additional stabilizer. Accordingly, in another embodiment of the invention, the detergent or detergent is characterized in that it contains at least one further stabilizer. Accordingly, the drug contains at least two compounds, which leads to stabilization of enzymes, particularly proteases, contained in the drug. These compounds preferably work synergistically. That is, the stability effect achieved by the two compounds exceeds the sum of the two individual stability effects. In a preferred embodiment, the stabilizer is one or more polyols, in particular glycerol or 1,2-ethylene glycol, antioxidants, lactates or one or more lactate derivatives or combinations thereof. is there. Also preferred are one or more enzyme stabilizing compounds and / or inhibitory compounds disclosed in international patent applications WO07 / 113241A1 or WO02 / 008398.

  The protease that is stabilized and / or reversibly inhibited according to the invention is preferably a serine protease, in particular a subtilase, particularly preferably a subtilisin.

  Examples of such proteases include subtilisin BPN ′ and Carlsberg, protease PB92, subtilisins 147 and 309, alkaline protease from Bacillus lentus, subtilisin DY and subtilase, although not strictly classified as subtilisin, the enzyme thermitase, Proteinase K or proteinases TW3 and TW7 are included. Subtilisin Carlsberg is available in further developed form under the trade name Alcalase® from Novozymes A / S (Bagsvaerd, Denmark). Subtilisins 147 and 309 are commercially available from Novozymes under the trade names Esperase® and / or Savinase®. Protease variants treated under the title BLAP® are derived from the protease obtained from Bacillus lentus DSM 5483.

  Further proteases include, for example, under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase®, Kannase® and Ovozymes®, Enzymes available from Novozymes, Purafect (R), Purafect (R) OxP and Properase (R) under the trade name Genencor, under the trade name Protosol (R), Enzymes available from Advanced Biochemicals Ltd. (Thane, India) under the trade name Wuxi®, enzymes available from Wuxi Snyder Bioproducts Ltd. (China), Proleather® and Protease P (registered) Under the trade name of “Trademark” and enzymes available from Amano Pharmaceutical Co., Ltd. (Nagoya, Japan) and enzymes available from Kao Corporation (Tokyo, Japan) under the trade name of Proteinase K-16.

  Surprisingly, it has been found that such proteases are particularly well stabilized and / or reversibly inhibited by the compounds described herein. Furthermore, even certain variants of proteases (ie including the above variants) are stabilized in a particularly advantageous manner by these compounds. Such protease variants are part of the subject of the invention described below.

  The stabilizing and / or reversibly inhibiting protease of the present invention may be a wild type enzyme or a protease variant. The term “wild-type enzyme” is understood to mean an enzyme that is present in and can be separated from naturally occurring organisms and / or natural habitats. However, enzymes are variable and can be altered in a way that is targeted to some degree to adapt their properties specifically to the intended use or to affect their catalytic activity. These changes often occur due to changes in the amino acid sequence of the enzyme. Such changes can occur in a targeted manner, either directionally or randomly, eg, by a random mutation process. Enzyme variants are understood to refer to enzymes made from a starting strain, such as a wild-type enzyme, for example by changes in the amino acid sequence. Changes in the amino acid sequence preferably occur through mutations by allowing amino acid substitutions, deletions, insertions or combinations thereof to be made. The introduction of such mutations into proteins is state of the art and is well known to those skilled in the art of enzyme technology. Essentially all enzymes can be changed by this method. According to the invention, protease variants are preferred. These are created by making amino acid substitutions, deletions, insertions or combinations thereof from a starting protease, such as a wild-type protease, by changing the amino acid sequence. However, the starting protease need not be a naturally occurring wild-type protease. Proteases known from the prior art that have already been changed may be further developed and may thus function again as starting proteases to create further protease variants.

  Thus, for example, all the proteases described above can be used in the agents of the present invention without change and may be stabilized by the compounds described herein. However, they can also mean starting enzymes for variants contained in the agents of the invention and stabilized by the compounds described herein.

Furthermore, the starting enzyme of the wild-type enzyme and / or the following variant is preferred for all proteases and / or variants described herein:
-Alkaline protease (BPN ') from Bacillus amyloliquefaciens,
-Alkaline protease from Bacillus licheniformis (Subtilisin Carlsberg)
-Alkaline protease PB92,
Subtilisin 147 and / or subtilisin 309 (savinase),
-An alkaline protease from Bacillus lentus, preferably Bacillus lentus DSM 5483,
-An alkaline protease (DSM 11233) from Bacillus alcalophilus,
An alkaline protease from Bacillus gibsonii (DSM 14391) or an alkaline protease at least 70% identical to the former,
An alkaline protease from Bacillus sp. (DSM 14390) or an alkaline protease at least 98.5% identical to the former,
An alkaline protease from Bacillus sp. (DSM 14392) or an alkaline protease at least 98.1% identical to the former,
-Alkaline protease from Bacillus gibsonii (DSM 14393) or an alkaline protease at least 70% identical to the former.

  Thus, in another embodiment of the invention, the detergent or detergent is obtained from the starting protease by at least one change in the amino acid, such as an amino acid substitution, insertion or deletion, and at least 90%, preferably at the amino acid level. At least 92.5%, particularly preferably at least 95%, most particularly preferably at least 97.5% are characterized by proteases that are identical to the starting protease.

  Those skilled in the art are well aware of how to perform and create sequence comparisons, so-called alignments, from the field of enzyme technology. For example, identity values or homology values for comparison sequences are determined by such sequence comparisons. Such a comparison is made by assigning similar sequences in the nucleotide or amino acid sequence of the protein in question to each other. This is known as homologization. The sequence listed for each position is known as an alignment. In analyzing the nucleotide sequence, both complementary strands and all three possible reading frames must be considered. Similarly, the degeneracy of genetic information and the use of organism-specific codons (codon usage) are considered. On the other hand, the alignment is created by a computer program using, for example, the FASTA or BLAST algorithm. This technique is described, for example, by D. J. Lipman and W. R. Pearson in Science, 227, pp. 1435-1441 (1985).

  A collection of all matching arrangements in the sequences to be compared in this way is known as a consensus sequence.

  Such a comparison allows for a reference to the similarity or homology of the sequences being compared to each other. This is indicated by the percent identity, i.e., the percentage of identical nucleotide or amino acid residues at the same position and / or corresponding positions in the alignment. In the broader term homology, this value includes conserved amino acid substitutions. In that case, we call the similarity rate. Such a reference may be made to the entire protein or gene or just individual regions.

  Homologous regions of different proteins are determined by amino acid sequence matches. They are also characterized by the same function. This ranges to a perfect match in a very small region, the so-called box, which consists of only a few amino acids and usually performs the function essential for overall activity. The function of the homologous region is understood to be a very subordinate function of the function exerted by the complete protein, such as the formation of individual hydrogen bridge bonds to generate a substrate complex or transition complex.

  Such sequence comparison and / or alignment is also particularly useful for finding corresponding positions in different molecules. For example, in an alignment of different enzymes, each sequence may have, for example, a different full length or a different region and / or subsequence, or if additional amino acids and / or nucleotides are present in the sequence. Even the position in each amino acid sequence or nucleic acid sequence that match each other can be determined. Therefore, a matching position in the secondary sequence can be specifically assigned to a specific position in the primary sequence, and it is sufficiently possible that the matching position is located in a different region in the molecule. Furthermore, different amino acid residues may be present at matching positions. Thus, for such configuration comparison and / or position determination, which position is relevant and which enzyme is used as starting material, ie which measurement method is used as the basis for position determination. It is specifically mentioned whether it is done.

  An alkaline protease from Bacillus lentus DSM 5483 disclosed in an unexamined international patent WO91 / 02792A1 and referred to in this patent application (referred to as an alkaline protease from Bacillus lentus in this patent application) The amino acid sequence of the mature protein is used for position determination.

  In another embodiment of the invention, the detergent or detergent is characterized in that the protease is obtained from the starting protease by at least one change in amino acid. The change is an amino acid substitution or insertion in the amino acid sequence region sequenced at positions 95 to 103 of the alkaline protease from Bacillus lentus in the alignment.

  Such protease variants are particularly preferably at positions 95, 96, 97, 98, 99, 100, 101, 102 and / or 103, most particularly preferably at positions 97 and 98 and / or positions 99 and 100. A variant by insertion of one or more single amino acids between.

  In another embodiment of the invention, the protease is in alignment with the alkaline protease 3, 4, 36, 42, 43, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104 from Bacillus lentus. 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255 and 268 Detergents or detergents are characterized in that they are derived from the starting protease by at least one change by substitution, insertion or deletion of a sequenced amino acid.

  One or more amino acid changes at the following positions compared to the starting molecule are particularly preferred: 3, 4, 43, 61, 188, 193, 199, 211, 224, 250 and 253 (alkaline from Bacillus lentus One or more amino acid substitutions of X3T, X41, X43V, X61A, X188P, X193M, X1991, X211L, X211D, X211E, X211G, X211N or X211Q, X224V, X250G and / or X253N It is. This protease is in particular a mutant by point mutation at position 211, preferably a mutant by substitution of a single amino acid at this position, particularly preferably a mutant by amino acid substitution of X211L. The positional information also refers to the amino acid residue located at the position of the alkaline protease from Bacillus lentus in the alignment.

  In addition to proteases, the agents of the present invention may contain one or more other enzymes, particularly from the following group: one or more additional proteases, amylases, hemicellulases, cellulases, lipases And oxidoreductase. The amylase is preferably α-amylase. The hemicellulase is preferably β-glucanase, pectinase, pullulanase and / or mannanase. The cellulase is preferably a cellulase mixture of single component cellulases, preferably and / or predominantly endoglucanase and / or cellobiohydrolase. The oxidoreductase is preferably an oxidase, in particular a choline oxidase or a perhydrolase.

  The agents of the present invention preferably contain at least one complexing agent and / or builder substance. The builder is in particular a zeolite builder and / or a nonionic surfactant, which is preferably a hydroxy mixed ether and / or an optical brightener, the optical brightener being a diphenyl compound, in particular distyryl-biphenyl. Derivatives and / or stilbene-triazine derivatives.

Example 1: Residual protease activity test in the presence of inhibitors In order to confirm that the compounds described below have a protease activity inhibitory effect, Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1) is present in the presence of these compounds. Measured below.

The substrates succinyl-alanine-alanine-proline-phenylalanine-paranitroanilide (AAPFpNA; Bachem L-1400) and 5 × 10 ⁻⁹ and / or 1 × 10 ⁻⁸ M protease were added to 100 mM Tris buffer. Thereafter, the test compounds listed in Table 1 were added to a final concentration of 10 mM. Each was dissolved in anhydrous DMSO and the corresponding control with the same amount of DMSO without each compound was used to correct the effect of DMSO on enzyme activity. The batch was incubated for 5 minutes at pH 8.6, 25 ° C. 1 U corresponds to 1 μmol of substrate that is degraded per minute.

The following compounds were tested in this way.
V1: 2-[[(3-carboxyphenyl) amino] carbonyl] benzoic acid V2: 2-[[(2-carboxybenzoyl) amino] benzoic acid V3: 2-[[(4-carboxyphenyl) amino] carbonyl] Benzoic acid V4: 2- (4-carboxybenzoyl) benzoic acid V5: 4- (2-carboxybenzoyl) -1,2-benzenedicarboxylic acid

  All of these resulted in a residual protease activity of 50% or less. V1 is the largest of these and is the most suitable protease inhibitor and / or stabilizer. After that, V2, V3, V4 (about the same as V3) and V5 followed.

  Based on these results, these compounds are also suitable for stabilizing enzyme activity in protease-containing detergents and detergents.

Example 2: Stability test of protease-containing detergents and detergents on storage in the presence of protease inhibitors Liquid detergents containing the following compounds were prepared as basic formulations (all amounts given in weight percent): 0 .3-0.5% xanthan gum, 0.2-0.4% antifoam, 6-7% glycerol, 0.3-0.5% ethanol, 4-7% FAEOS, 24- 28% nonionic surfactant, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acid, 0.5 % HEDP, 0-0.4% PVP, 0-0.05% optical brightener, 0-0.001% colorant, the balance demineralized water.

  This formulation was mixed with the inhibitor to be tested and 1,275,000 HPU / L B. lentus alkaline protease F49. Protease activity shown in HPU (Henkel protease unit) is described in a paper by van Raay, Saran and Verbeek: Tenside [Surfactants] (1970), Vol. 7, pp. 125-132, “For determination of the proteolytic activity in enzyme concentrates and Measurement was performed according to the description of “enzyme-containing washing, cleaning agents and dishwashing agents”.

  They were stored for various periods in a 30 ° C. hermetically sealed container.

  For analysis, the initial value of proteolytic activity of each drug was compared with the measured value after storage. The higher the activity remaining after storage, the better the inactivation of the protease contained therein during storage, and each compound more suitable as a stabilizer of the present invention.

  All the compounds tested had clearly a stabilizing effect.

Claims (32)

A detergent or detergent containing a protease and a compound having the following general structural formula.

[Where:
(A) X represents a carbonyl group (C═O) or an acid amide group (NHCO),
(B) R1, R2, R3, R4 and R5 (in ring 1) are hydrogen (H), carboxyl group (COOH), methyl group so that at least one carboxyl group (COOH) is present in the ring; (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen,
(C) R6, R7, R8, R9 and R10 (in ring 2) are hydrogen (H), carboxyl group (COOH), methyl group such that at least one carboxyl group (COOH) is present in the ring (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen, and
(D) Optionally, the two groups R1 to R10 (A) and (B) may be ortho to each other, wherein (A) is an essential component as described in (b) and / or (c) A carboxyl group (COOH) or optionally an additional carboxyl group, and (B) is a hydroxymethyl group, which are present as the above groups or optionally as CH ₂ —O—CO— groups, Represents a five-membered lactone with the carbon atoms of the ring it contains. ]   Most preferably, the stabilizing compound has 1 to 3 carboxyl groups, preferably 1 or 2 carboxyl groups, including the carboxyl groups bonded to the lactone according to (d) for each of the two aromatic rings. The cleaning or cleaning agent according to claim 1, which has 2 or 3 carboxyl groups on both rings simultaneously. The stabilizing compound has an inhibition constant (K _i ) of 0.01 to 10 mM, preferably 0.1 to 5, particularly preferably 0.5 to 2, with respect to the protease it contains. The cleaning agent or cleaning agent as described in 1. The cleaning or cleaning agent according to any one of claims 1 to 3, wherein the stabilizing compound is selected from one of the following stabilizers:

  The cleaning agent or cleaning agent according to any one of claims 1 to 4, which is mainly solid.   The cleaning agent or cleaning agent according to any one of claims 1 to 4, which is mainly liquid, paste-like or gel-like.   Protease is present in an amount of 2 μg to 20 mg per gram of agent, preferably 5 μg to 17.5 mg per gram of agent, particularly preferably 20 μg to 15 mg per gram of agent, most particularly preferably 50 μg to 10 μg per gram of agent. The cleaning agent or cleaning agent in any one of 1-6.   Washing according to any of the preceding claims, wherein the stabilizer is included in an amount of up to 50 mg, preferably up to 10 mg, particularly preferably up to 7 mg, most particularly preferably up to 5 mg per gram of agent. Agent or detergent.   The molar ratio of stabilizer to protease is from 1: 1 to 1000: 1, in particular from 1: 1 to 500: 1, particularly preferably from 1: 1 to 100: 1, most particularly preferably from 1: 1 to 20: 1. The cleaning agent or cleaning agent according to any one of claims 1 to 8. Stabilizer is included in an amount of 0.01-100 × K _i (based on the contained protease), preferably 0.1-10 × K _i , particularly preferably 1-5 × K _i , Item 10. A cleaning agent or a cleaning agent according to any one of Items 1 to 9.   11. The cleaning or cleaning agent according to any one of claims 1 to 10, wherein the protease is a serine protease, preferably a subtilase, particularly preferably a subtilisin.   The protease is obtained from the starting protease by at least one change, such as an amino acid substitution, insertion or deletion, and is at least 90% consistent, preferably at least 92.5% consistent with the starting protease at the amino acid level, particularly preferably 12. A cleaning or cleaning agent according to any of claims 1 to 11, which is at least 95% consistent, most preferably at least 97.5% consistent.   The protease is obtained by at least one amino acid change, wherein the change is an amino acid substitution or insertion in the region of the amino acid sequence located at positions 95 to 103 of the alkaline protease from Bacillus lentus in the alignment. The cleaning agent or cleaning agent in any one of 12.   The protease is an alkaline protease from Bacillus lentus 3, 4, 36, 42, 43, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, in alignment. 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255, and 268, substitutions or insertions of amino acids 14. A cleaning or cleaning agent according to any of claims 1 to 13, obtained from the starting protease by at least one change such as a deletion.   15. A cleaning or cleaning agent according to any of claims 1 to 14, containing at least one additional stabilizer.   16. Stabilizer is one or more polyols, in particular glycerol or 1,2-ethylene glycol, antioxidants, lactates or one or more lactic acid derivatives or combinations thereof. Cleaning or cleaning agent.   17. In particular, comprising one or more additional enzymes from the following group: one or more additional proteases, amylases, hemicellulases, cellulases, lipases and oxidoreductases. The cleaning agent or cleaning agent in any one.   The cleaning agent or detergent according to claim 17, wherein the amylase is α-amylase.   The cleaning or cleaning agent according to claim 17 or 18, wherein the hemicellulase is β-glucanase, pectinase, pullulanase and / or mannase.   20. A cleaning or cleaning agent according to any of claims 17 to 19, wherein the cellulase is a cellulase mixture or a single component cellulase, preferably and / or mainly an endoglucanase and / or a cellobiohydrolase.   21. A cleaning or cleaning agent according to any of claims 17 to 20, wherein the oxidoreductase is an oxidase, in particular a choline oxidase or a perhydrolase.   The cleaning or cleaning agent according to any one of claims 1 to 21, comprising at least one complexing agent and / or builder substance.   23. A cleaning or cleaning agent according to claim 22, wherein the builder is a zeolite builder.   The cleaning agent or cleaning agent in any one of Claims 1-23 containing a nonionic surfactant.   25. The agent of claim 24, wherein the nonionic surfactant is a hydroxy mixed ether.   The cleaning agent or cleaning agent according to any one of claims 1 to 25, which contains an optical brightener.   27. A cleaning or cleaning agent according to claim 26, wherein the optical brightener is a diphenyl compound, in particular a distyryl-diphenyl derivative and / or a stilbene-triazine derivative. Use of a compound of the following general structural formula as a reversible inhibitor of a protease within a detergent or detergent formulation.

[Where:
(A) X represents a carbonyl group (C═O) or an acid amide group (NHCO),
(B) R1, R2, R3, R4 and R5 (in ring 1) are hydrogen (H), carboxyl group (COOH), methyl group so that at least one carboxyl group (COOH) is present in the ring; (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen,
(C) R6, R7, R8, R9 and R10 (in ring 2) are hydrogen (H), carboxyl group (COOH), methyl group such that at least one carboxyl group (COOH) is present in the ring (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen, and
(D) Optionally, the two groups R1 to R10 (A) and (B) may be ortho to each other, wherein (A) is an essential component as described in (b) and / or (c) A carboxyl group (COOH) or optionally an additional carboxyl group, and (B) is a hydroxymethyl group, which are present as the above groups or optionally as CH ₂ —O—CO— groups, Represents a five-membered lactone with the carbon atoms of the ring it contains. ] A cleaning or cleaning method for activating a protease, wherein the protease is inhibited and / or stabilized by a compound of the following general structural formula:

[Where:
(A) X represents a carbonyl group (C═O) or an acid amide group (NHCO),
(B) R1, R2, R3, R4 and R5 (in ring 1) are hydrogen (H), carboxyl group (COOH), methyl group so that at least one carboxyl group (COOH) is present in the ring; (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen,
(C) R6, R7, R8, R9 and R10 (in ring 2) are hydrogen (H), carboxyl group (COOH), methyl group such that at least one carboxyl group (COOH) is present in the ring (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen, and
(D) Optionally, the two groups R1 to R10 (A) and (B) may be ortho to each other, wherein (A) is an essential component as described in (b) and / or (c) A carboxyl group (COOH) or optionally an additional carboxyl group, and (B) is a hydroxymethyl group, which are present as the above groups or optionally as CH ₂ —O—CO— groups, Represents a five-membered lactone with the carbon atoms of the ring it contains. ]   30. The cleaning or cleaning method according to claim 29, wherein the cleaning agent or cleaning agent according to any one of claims 1 to 27 is used.   28. Use of a cleaning or cleaning agent according to any of claims 1 to 27 for cleaning and / or cleaning textiles and / or hard surfaces. Use of a protease and a compound of the following general structural formula for the manufacture of a detergent or detergent.

[Where:
(A) X represents a carbonyl group (C═O) or an acid amide group (NHCO),
(B) R1, R2, R3, R4 and R5 (in ring 1) are hydrogen (H), carboxyl group (COOH), methyl group so that at least one carboxyl group (COOH) is present in the ring; (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen,
(C) R6, R7, R8, R9 and R10 (in ring 2) are hydrogen (H), carboxyl group (COOH), methyl group such that at least one carboxyl group (COOH) is present in the ring (CH ₃ ), ethyl group (C ₂ H ₅ ), hydroxyl group (OH), hydroxymethyl group (CH ₂ OH), amino group (NH ₂ ) and / or halogen, and
(D) Optionally, the two groups R1 to R10 (A) and (B) may be ortho to each other, wherein (A) is an essential component as described in (b) and / or (c) A carboxyl group (COOH) or optionally an additional carboxyl group, and (B) is a hydroxymethyl group, which are present as the above groups or optionally as CH ₂ —O—CO— groups, Represents a five-membered lactone with the carbon atoms of the ring it contains. ]